Objective: This study aimed to investigate the effect and significance of a binding protein-2 (Gab2)-Akt-ARK5 signaling pathway on the invasion of glioma cells. Methods: Immunohistochemical methods were used to detect the expressions of Gab2 and ARK5 in 45 cases of glioma tissue. siRNA plasmid was used to transfect LN-229 cells, and western blot was performed to analyze the protein expressions of Gab2 and ARK5. In vitro Matrigel invasion assay was conducted to detect variations in the invasiveness of transfected cells. Western blot was also conducted to analyze the protein phosphorylation of Akt and ARK5 in the cells transfected with Gab2 plasmid. Results: Immunohistochemical assay revealed that the expressions of ARK5 and Gab2 in glioma cells were positively correlated, and both expressions were higher in high-grade glioma (WHO grade III, IV) than in low-grade glioma (WHO grade I, II). LN-229 cells transfected with ARK5 plasmid, Gab2 plasmid, ARK5 and Gab2 plasmid, and control plasmid were named si ARK5/LN-229, siGab2/LN-229, siARK5 and siGab2/LN-229, and SCR/LN-229, respectively. After transfection was performed, the protein expressions of ARK5 and Gab2 were respectively decreased in siARK5/LN-229 and siGab2/LN-229. The protein expressions of ARK5 and Gab2 in siARK5 and siGab2/LN-229 were also respectively decreased. After ARK5 or Gab2 was downregulated, the number of glioma cells, which invaded and penetrated Matrigel, was decreased (P<0.01). The number of glioma cells also decreased significantly after ARK5 and Gab2 were downregulated. The phosphorylation of Akt and ARK5 in siGab2/LN-229 cells was decreased after these cells were stimulated by insulin-like growth factor-1. Conclusion: The silencing of ARK5 or Gab2 impaired glioma cell invasiveness. The decreased protein expression of Gab2 inhibited the phosphorylation of Akt and ARK5. These results suggested that the Gab2-Akt-ARK5 signaling pathway could be relevantly involved in glioma cell invasion.